The solution of platelet factors released by the activated thrombocytes (also known as "platelets") is often denoted by the expression "activated-thrombocyte supernatant".
Activated-thrombocyte supernatants are currently used as starting materials for the purification of biological molecules, such as, for example: platelet-derived growth factor, transforming growth factor-.beta., basic fibroblast growth factor, platelet factor 4, platelet-derived endothelial growth factor, heparin-binding epidermal growth factor, insulin-like growth factor 1, connective tissue activating peptide III, .beta.-thromboglobulin, epidermal growth factor, plasminogen, Von Willsbrand factor, fibrinogen, serotonin, histamine, adenosine di- and triphosphate, fibronectin, vitronectin, factor XIII, proteolytic or glycolytic enzymes, or the metabolites of arachidonic acid (see the review, Inflammation and Repair, H. L. Wong & S. M. Wahl, in "Peptide Growth Factors and their Receptors", p. 510, edited by Sporn & Roberts, Springer-Verlag, Berlin; Platelets and Response to Injury, R. A. Terkeltaub & M. H. Ginsberg, in "The Molecular and Cellular Biology of Wound Repair", p. 38, edited by Clark & Henson, Plenum Press, New York).
Certain purification methods are known and were able to form the subject of patent filings. Mention may especially be made of: European Patent No. 89100148.9 relating to the purification of transforming growth factor-.beta. and U.S. Pat. No. 4,479,896 relating to the purification of platelet-derived growth factor.
Activated-thrombocyte supernatants (or a fraction of these supernatants) can be used in therapy, for example for their healing activity (D. Knighton et al., Ann. Surg. (1982) 196, 379-388; D. M. Carter et al., in: Growth Factors and Other Aspects in Wound Healing, p. 303-317, edited by Barbul, Pines, Cadwell and Hunt, Alan R. Liss Inc., New York, 1988; U.S. Pat. No. 4,760,131; Patent WO 88/03409; Patent WO 86/03122; Patent WO 89/05656), or in cosmetology, for example for their beneficial effect in controlling alopecia (Patent WO 90/07931).
Platelet factors contained in activated-thrombocyte supernatants can be used especially in the treatment of ulcers.
The methods described to date for the preparation of thrombocytic supernatants are lengthy (minimum duration of approximately 1 hour 30 minutes), tedious (because they require transfers from tube to tube, getting the centrifuging balanced, pipettings) and virtually impossible to automate and they present risks of contamination of the product by its environment (transfers of liquids in open-necked vessels) or of the technician by the product being handled, which is potentially infected by transmittable germs.
In fact, these preparation methods begin with the isolation of the thrombocytes from the blood by successive centrifugings. The thrombocytes are then washed by centrifuging to remove the plasma (Isolation of Platelets, in: Methods in Enzymology, Section I, pp. 3-27, Vol. 169, edited by Hawiger, Academic Press, London, 1989; Patent WO 86/03122, U.S. Pat. No. 4,760,131). The purified thrombocytes are then suspended in the presence of an activator. Many activators are known (Platelets and Response to Injury, R. A. Terkeltaub & M. H. Ginsberg, in "The Molecular and Cellular Biology of Wound Repair", pp. 35-55, edited by Clark a Henson, Plenum Press, New York). Three types of activators are distinguished: strong activators which are capable of inducing secretion from all the granules (thrombin, collagen, calcium ionophore A23187), intermediate activators such as thromboxane A2, ADP in the presence or absence of calcium ions, or adrenalin, and weak activators which do not induce granular secretion (serotonin). The most used activator is thrombin. Other activation methods use physical methods for the lysis of thrombocytes by successive freezings/defrostings or by ultrasound. The thrombocytic supernatant is clarified by centrifuging and/or filtration in order to remove the thrombocytic membranes and other insoluble components.
In addition, in conventional techniques, the contaminants of plasma origin are difficult to remove and the successive washings which are targeted at removing these plasma contaminants lead to a reduction in the yield of thrombocytic molecules by loss of thrombocytes or by partial activation of the latter. Finally, the plasma in which the thrombocytes are suspended is difficult to recover for another use.
The present invention makes it possible to solve the problems described and which are inherent in the current methods for the preparation of platelet supernatants, such as risks of contamination, loss of yield and the need for lengthy and problematic manipulations.